title: Phosphorylation regulates kinase and microtubule binding activities of the budding yeast chromosomal passenger complex in vitro.
abstract: The chromosomal passenger complex (CPC) is a key regulator of mitosis in eukaryotes. It comprises four essential and conserved proteins known in mammals/yeasts as Aurora B/Ipl1, INCENP/Sli15, Survivin/Bir1, and Borealin/Nbl1. These subunits act together in a highly controlled fashion. Regulation of Aurora B/Ipl1 kinase activity and localization is critical for CPC function. Although regulation of CPC localization and kinase activity in vivo has been investigated elsewhere, studies on the complete, four-subunit CPC and its basic biochemical properties are only beginning. Here we describe the biochemical characterization of purified and complete Saccharomyces cerevisiae four-subunit CPC. We determined the affinity of the CPC for microtubules and demonstrated that the binding of CPC to microtubules is primarily electrostatic in nature and depends on the acidic C-terminal tail (E-hook) of tubulin. Moreover, phosphorylation of INCENP/Sli15 on its microtubule binding region also negatively regulates CPC affinity for microtubules. Furthermore, we show that phosphorylation of INCENP/Sli15 is required for activation of the kinase Aurora B/Ipl1 and can occur in trans. Although phosphorylation of INCENP/Sli15 is essential for activation, we determined that a version of the CPC lacking the INCENP/Sli15 microtubule binding region (residues Glu-91 to Ile-631) is able to form an intact complex that retains microtubule binding activity. Thus, we conclude that this INCENP/Sli15 linker domain plays a largely regulatory function and is not essential for complex formation or microtubule binding.
authors.affiliation: Department of Molecular and Cell Biology University of California, Berkeley, California 94720, USA.
authors.forename: Anthony
authors.initials: A
authors.lastname: Cormier
authors.forename: David G
authors.initials: DG
authors.lastname: Drubin
authors.forename: Georjana
authors.initials: G
authors.lastname: Barnes
mesh_terms: D064127:Aurora Kinases; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism*; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism*; D008870:Microtubules / Q000235:genetics / Q000378:metabolism*; D046912:Multiprotein Complexes / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation / Q000502:physiology; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D047908:Intracellular Signaling Peptides and Proteins; D008869:Microtubule-Associated Proteins; D046912:Multiprotein Complexes; D029701:Saccharomyces cerevisiae Proteins; C480084:Sli15 protein, S cerevisiae; D064127:Aurora Kinases; C576961:IPL1 protein, S cerevisiae; D017346:Protein Serine-Threonine Kinases
keywords: Cancer Biology; Cell Biology; Cell Cycle; Enzyme Purification; Kinase; Mitotic Spindle; Tubulin